Method and device for detecting feline immunodeficiency virus

ABSTRACT

A method and device for determining a feline immunodeficiency virus infection or vaccination in an animal. The method includes contacting a biological sample from a felid with various FIV polypeptides and determining the binding of antibodies in the sample to the polypeptides. The determination of whether an animal is infected with FIV or has been vaccinated against FIV can be determined by measuring the animal&#39;s immune response to an FIV env polypeptide. A device for detecting FIV antibodies is provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/584,715, filed Jun. 30, 2004.

FIELD OF THE INVENTION

The invention is related to the detection of antibodies directed toFeline Immunodeficiency Virus.

BACKGROUND OF THE INVENTION

Feline immunodeficiency virus (FIV), formerly called felineT-lymphotrophic lentivirus, was first isolated in 1986 from a largemultiple cat household in Petaluma, Calif. (Pederson et al., Science(1987) 235:790). FIV infects cats to produce an AIDS-like syndrome.Although FIV is morphologically and pathologically similar to the humanimmunodeficiency virus (HIV), it has been shown to be antigenicallydistinct from HIV. Like HIV, once a cat becomes infected with FIV, thedisease progresses from a primary infection (viraemia, fever, generallymphadenitis) to a lengthy asymptomatic phase, followed by severeimpairment in immune function caused by a reduction in CD4 lymphocytes,and resulting in heightened susceptibility to secondary infections andultimately death.

FIV has been classified as a member of the subfamily Lentiviridae in thefamily Retroviridae, the family that includes human and simianimmunodeficiency viruses, equine infectious anaemia, maedi visna ofsheep and caprinearthritis encephalitis viruses (CAEV). The genome ofFIV is organized like other lentiviruses with three long open readingframes corresponding to gag, pol and env (Talbott et al., Proc. Natl.Acad. Sci. (1989) 86:5743; Olmsted et al., Proc. Natl. Acad. Sci. (1989)86:2448). The gag gene codes for the major structural components of thevirus, the env gene codes for the envelope glycoprotein, and the polgene codes for the polymerase protein.

The gag gene is expressed as a 55 kD polyprotein that is processed intothree subunits: a p15 matrix protein, a p24 capsid protein, and a p10nucleocapsid protein. The pol gene encodes three proteins: the protease,reverse transcriptase and a p14.6 protein of unknown function.Autoprocessing by the protease portion of the gene gives rise to allthree proteins of the pol region. Additionally, the protease isresponsible for the processing of the gag precursor. The pol gene isexpressed as a gag-pol fusion protein. The envelope gene is expressed asa 160 kD glycoprotein, gp160. The antigenicity of the FIV core proteinsis similar to other lentiviruses.

Several independent viral isolates have been prepared across the world,and a certain number of studies have been carried out in order todemonstrate the structure of the isolated strains: the American strainPetaluma, Talbott et al. Natl. Acad. Sci. USA, 1989, 86, 5743-5747;Philipps et al., J. Virol., 1990, 64, 10, 4605-4613, the Japanesestrains (the TM1 and TM2 strains), Miyazawa et al., Arch. Virol., 1989,108, 59-68, and the Swiss isolates (FIVZ1 and FIVZ2), Morikawa et al.,Virus Research, 1991, 21, 53-63.

The nucleotide sequences of three proviral clones derived from AmericanFIV isolates (Petaluma strain) have been described (clones FIV34TF10,FIV14 and isolate PPR) (Olmsted, et al. 1989; Philipps et al., 1990;Talbott et al., 1989) and compared with two Swiss isolates (Morikawa etal. 1991). This comparison led Morikawa et al. to specify the presenceof certain conserved regions and certain variable regions within the envgene of FIV. French strains have also been isolated (strains Wo and Me)(Moraillon et al., 1992, Vet. Mic., 31, 41-45).

The virus replicates optimally in blood mononuclear cells and has atropism for T-lymphocytes, peritoneal macrophage, brain macrophage andastrocytes. In common with other retroviruses, the genetic material ofFIV is composed of RNA and the production of a DNA copy of the viral RNAis an essential step in the replication of FIV in the host. This steprequires the enzyme reverse transcriptase that is carried into the hostby the invading virus. The DNA version of the viral genome is insertedinto the genetic material of infected host cells in which it continuesto reside as a provirus. This provirus is replicated every time the celldivides and can code for the production of new virus particles. Cellsinfected with FIV remain infected for the duration of their lifespan.

The virus appears to be spread naturally by horizontal transmission,predominantly by bite wounds from an infected cat as these animals shedappreciable amounts of virus in saliva (Yamamoto et al., Am. J. Vet.Res. 1988, 8:1246). Vertical transmission has been reported, but israre.

Current diagnostic screening tests for FIV infection detect serumantibody (Ab) to FIV. Virus detection kits are also available but not asprevalent. A number of diagnostic tests are available to determine thepresence of FIV antibody in infected animals. For example, PetChek® FIVAb test kit and the SNAP® Combo FeLV Ag/FIV Ab test kit (IDEXXLaboratories, Westbrook, Me.) are immunoassay based diagnostic tests forFIV infection.

Detecting FIV infection is becoming increasingly important as studiesreveal FIV infection is widespread worldwide. As vaccines have beendeveloped in attempt to combat the disease, it is even more important tobe able to detect the effectiveness of a vaccine and to discriminatebetween vaccinated cats versus naturally infected cats.

SUMMARY OF THE INVENTION

In one aspect, the invention is directed to novel FIV polypeptides. Inanother aspect, the invention provides a method for determining whethera felid has been vaccinated against FIV or is naturally infected withFIV by determining the felid's immune response to an FIV polypeptide,such as an FIV env polypeptide.

In another aspect, the invention is directed to a method ofdistinguishing among animals that have been naturally infected with FIVversus animals that have not been infected or have been vaccinatedagainst an FIV infection. The method includes contacting a biologicalsample from an animal with a polypeptide that does not substantiallybind to an FIV antibody that is a significant component of the animal'simmune response to an FIV vaccine. FIV antibodies in the sample thatsubstantially bind to the polypeptide are detected. It is determinedthat the animal is naturally infected by correlating a positive resultin the detecting step to a natural infection and it is determined thatthe animal has been vaccinated or not infected by correlating a negativeresult to a vaccination or no infection. The polypeptide may be derivedfrom FIV env.

In a further aspect, the invention is directed to a method ofdetermining whether a cat has been vaccinated against FIV or isnaturally infected with FIV. The method includes (a) detecting, before aperiod a time following vaccination sufficient for certain FIVantigen-specific antibodies raised in response to the vaccine to be notdetected, whether the cat has antibodies against an FIV peptide; (b)detecting, after a period of time following vaccination sufficient forcertain FIV antigen-specific antibodies raised in response to thevaccine to be not detected, whether the cat has antibodies against anFIV polypeptide; (c) determining that the animal has been vaccinated bydetecting antibodies in step a but not in step b; and (d) determiningthat the animal is naturally infected by detecting antibodies in steps aand b.

The invention also provides for a method of determining whether a cathas not been infected by FIV or has been vaccinated against FIV. Themethod includes analyzing a biological sample from the cat to detectantibodies against a polypeptide derived from FIV, and determining thatthe animal has not been infected or has been or vaccinated by notdetecting such antibodies.

In yet another aspect, the invention provides a method of determiningwhether or not a cat has been vaccinated for FIV or naturally infectedwith FIV. The method includes providing a test device comprising apolypeptide, obtaining a blood sample from a cat, running the bloodsample on the test device, and reading the test device. A positiveresult indicates the cat has been naturally infected with FIV orvaccinated against FIV and a negative result indicates the cat has notbeen naturally infected with FIV and not vaccinated against FIV.

Still further, the invention is directed to a diagnostic device having adry porous carrier, a first detection reagent immobilized on the porouscarrier where the first detection reagent includes a protein thatcaptures FIV antibodies generated by a host in response to either anatural FIV infection or an FIV vaccination, and a second detectionreagent immobilized on the porous carrier wherein the second detectionreagent includes a protein that captures FIV antibodies generated by ahost in response to a natural FIV infection but does not substantiallycapture antibodies generated by the host in response to an FIVvaccination. The first detection reagent may be FIV p15 or p24 antigen,and the second detection reagent may be an FIV env protein.

DETAILED DESCRIPTION

Before describing the present invention in detail, a number of termswill be defined. As used herein, the singular forms “a,” “an”, and “the”include plural referents unless the context clearly dictates otherwise.

As used herein, the term “polypeptide” refers to a compound of a singlechain or a complex of two or more chains of amino acid residues linkedby peptide bonds. The chain(s) may be of any length. A protein is apolypeptide and the terms are used synonymously. Also included withinthe scope of the invention are functionally equivalent variants andfragments of FIV polypeptides. The polypeptide is capable of binding oneor more antibodies specific for the polypeptide.

Polypeptides derived from FIV include any region of the of the FIVproteome including for example, portions of the gag and env regions andmimitopes thereof. U.S. Pat. Nos. 5,648,209, 5,591,572, and 6,458,528,which are incorporated by reference herein in their entirety, describeFIV polypeptides derived from the FIV env and gag proteins. Thesepeptides, and others like them, from the env and gag proteins, aresuitable for use in the methods of the present invention An example of asuitable env polypeptide includes the following: CELGCNQNQFFCK [SEQ IDNO: 1]

SEQ ID NO:1 is the native FIV env sequence, amino acids 696-707, shownhere with a non-native N-terminal cysteine residue.

“Binding specificity” or “specific binding” refers to the substantialrecognition of a first molecule for a second molecule, for example apolypeptide and a polyclonal or monoclonal antibody, or an antibodyfragment (e.g. a Fv, single chain Fv, Fab′, or F(ab′)2 fragment)specific for the polypeptide.

“Substantial binding” or “substantially bind” refer to an amount ofspecific binding or recognizing between molecules in an assay mixtureunder particular assay conditions. In its broadest aspect, substantialbinding relates to the difference between a first molecule'sincapability of binding or recognizing a second molecule, and the firstmolecules capability of binding or recognizing a third molecule, suchthat the difference is sufficient to allow a meaningful assay to beconducted distinguishing specific binding under a particular set ofassay conditions, which includes the relative concentrations of themolecules, and the time and temperature of an incubation. In anotheraspect, one molecule is substantially incapable of binding orrecognizing another molecule in a cross-reactivity sense where the firstmolecule exhibits a reactivity for a second molecule that is less than25%, preferably less than 10%, more preferably less than 5% of thereactivity exhibited toward a third molecule under a particular set ofassay conditions, which includes the relative concentration andincubation of the molecules. Specific binding can be tested using anumber of widely known methods, e.g, an immunohistochemical assay, anenzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), ora western blot assay.

Animals infected with FIV are felids, which is to be understood toinclude all members of the order Felidae, including domestic cats,lions, tigers, jaguars, leopards, puma, ocelots, etc. As used herein,the terms “felid,” “cat” or “animal” is a reference to all felids.

A “biological sample” refers to a sample from an animal subjectincluding saliva, whole blood, serum, plasma or other sample known tocontain FIV antibodies.

An “antibody that is a significant component of an animal's immuneresponse to a FIV vaccine” refers to an antibody that is elicited as theresult of a vaccination with a FIV vaccine. These antibodies may beidentical to or similar to antibodies elicited as the result of anatural FIV infection. A successful vaccination produces a measurablelevel of the antibody that is a significant component of an animal'simmune response to the FIV vaccine.

Vaccines for FIV are described, for example, in U.S. Pat. Nos.6,667,295, 5,833,993, 6,447,993, 6,254,872 and 6,544,528, and publishedU.S. Patent Application 20040096460, each of which is incorporatedherein by reference in their entirety. U.S. Pat. Nos. 6,447,993 and6,254,872 describe vaccines that are prepared from cell free-viralisolates of different FIV subtypes or a combination of cell lines eachinfected with different prototype FIV virus from a different subtype.U.S. Pat. No. 5,833,933 describes vaccines containing DNA sequencesencoding FIV gag protein and FIV env protein. These vaccines include anexpression system for expressing the sequences. One available vaccine isFEL-O-VAX® FIV (Fort Dodge Animal Health, Overland Park, Kans.).

Biological samples from animals that have been vaccinated against an FIVinfection have the potential for producing a positive result in a testfor an FIV infection due to the presence of antibodies produced by theanimal in response to the vaccine. In one aspect, the invention providesfor a method of distinguishing between animals that have been naturallyinfected with FIV and animals that have not been infected or have beenvaccinated against an FIV infection. The method includes contacting abiological sample from the animal with a polypeptide derived from an FIVthat does not substantially bind to an antibody that is a significantcomponent of the animal's antibody response to an FIV vaccine.

In another aspect, the invention includes a method of determiningwhether a cat has not been infected by FIV and has not been vaccinatedagainst FIV. A biological sample from a cat is analyzed to detectantibodies against a polypeptide, derived from FIV env and/or gag. It isthen determined that the animal has not been infected and has not beenor vaccinated by determining the absence of such antibodies.

In some instances, during an initial phase following a vaccination, ananimal may temporarily (transiently) produce lower levels of certainantibodies to specific FIV polypeptides that are elements of a vaccine,as compared to those produced in response to a natural infection. Theseantibody levels taper off after a period of time to the point thatantibody to these polypeptides is not detected after the initial phase.Generally, this amount of time is about ten to twelve weeks, but willvary between species and individual subject animals. Transientantibodies are not a significant component of the animal's immuneresponse to the vaccine.

For example, the development of FIV antibodies in an animal against avaccine is dependent upon the vaccine. It has been found that animalstest seropositive for FIV antibodies against p24 (gag) about two to fourweeks after vaccination with the FEL-O-VAX® vaccine. However, animals sovaccinated do not generate persistent levels of antibodies against oneor more regions of the env protein, even though this protein wasincluded as an element of the vaccine. In contrast, naturally infectedanimals typically generate persistent levels of antibodies to both FIVgag and env proteins.

The differences in the immune response between animals that arevaccinated and animals that are naturally infected provide a means fordetermining whether an animal has been vaccinated or is naturallyinfected. Using the method of the invention, animals that have beennaturally infected with FIV can be distinguished from animals that havenot been infected or have been vaccinated against an FIV infection.Accordingly, the detection of the substantial binding between apolypeptide derived from FIV and an antibody that is not a significantcomponent of an animal's immune response to a vaccine can indicate anatural infection. The relative absence of such binding can indicatevaccination or no infection. In addition, a second, separate antibodycapture reagent can be included in the test that substantially binds toantibodies produced in response to vaccination and/or natural infection,such as p15 or p24 proteins. As such, various combinations of separatecapture reagents can lead to a determination of the vaccination and/orinfection status of the test subject.

For example, FIV gag proteins p15 and p24 may be immunogenic componentsof a killed whole virus FIV vaccine. It is expected that thesecomponents elicit a persistent antibody response when administered to ananimal. On the other hand, some vaccines may not include immunologicallysignificant quantities of FIV env protein or, this protein has beenaltered in the process of virus inactivation, or presentation of thisprotein by vaccination differs from that for natural infection to apoint where antibodies produced thereto, if any, are detected for aperiod of time less than antibodies to p15 and p24. Thus, while duringthe initial phase following vaccination, an animal may transientlyproduce low levels of such antibodies that bind to env proteins, anysuch antibody production declines over a period of time and is notdetected after about 12 weeks. In this example, the transiently producedantibodies are not a significant component of the animal's immuneresponse to the vaccine after a period of time.

Given that the production of detectable antibodies that are directedtoward specific FIV env polypeptides usually drops off after about 12weeks from completion of vaccination, in one aspect of the invention thebiological sample is obtained from the animal that has not received anFIV vaccine within about the prior 12 weeks. If the vaccination statusis unknown and the test indicates infection (based on a reaction withthe antibody capture protein), a retest after an additional 12 weeks canbe recommended.

Differences in the immune response between vaccinated animals andnaturally infected animals, such as specific antibody levels and/orkinetic parameters for antibody-antigen binding reactions (e.g.,affinities, avidities), should be considered in the design of an assayfor distinguishing between vaccinated and infected animals. Differencesin immune response can be significant such that even after the initialphase following a vaccination, an animal may persistently produce lowerlevels of antibodies to specific FIV polypeptides, and/or antibodieswith different binding properties as compared to those antibodiesproduced in response to a natural infection.

The method of the invention can be optimized in many ways and one ofskill in the art could simultaneously adjust the sample dilutions,reagent concentrations, incubation temperatures and times used in themethod to accomplish a differential detection of serum having antibodiesto an FIV infection or vaccination. For instance, at optimized sampledilution and other conditions for an immunoassay for antibodies tospecific polypeptides, samples from vaccinated animals may, for onespecific FIV polypeptide, give a negative assay result and samples frominfected animals will give a positive assay result. For a second FIVpolypeptide, both samples may give a positive result.

In one aspect of the invention, the proteins are immobilized on asuitable solid support. The biological sample is brought into contactwith the protein, to which the anti-FIV antibodies bind, if suchantibodies are present in the sample. The binding may be detected by anysuitable means, e.g., enzymes, radionuclides, particulates orfluorescent labels. In a suitable embodiment, the detection reagent canbe associated with a protein that is the same or similar to that whichis used to capture anti-FIV antibodies (if present).

In another aspect, the method is directed to a test device to determinewhether a cat has been vaccinated for FIV or naturally infected withFIV. A method of using the test device includes providing a test devicehaving an FIV env protein and a separate FIV gag protein. The device canbe used to test a biological sample from a cat by contacting the devicewith the biological sample. Upon reading the device, the detection ofthe binding of an antibody to the gag protein (a positive result on thegag protein) indicates the cat has been naturally infected with FIV orvaccinated against FIV. A concurrent positive result on the env proteinindicates natural infection (or, perhaps a transient post vaccinationresponse), while a concurrent negative result on the env proteinindicates vaccination. The following table summarizes the above: gagprotein env protein No vaccination or infection − − Vaccination + −Potential Recent Vaccination + + Infection + + Infection andVaccination + +

The polypeptides used in the invention contain at least six amino acids,usually at least nine amino acids, and more usually twelve or more aminoacids found within one of the natural FIV proteins and mimitopes andfunctionally equivalent variants thereof.

“Functional equivalent” or “Functionally equivalent” refers topolypeptides related to or derived from the native FIV envelope (env)and viral core (gag) polypeptide sequences where the amino acid sequencehas been modified by a single or multiple amino acid substitution,insertion, deletion, and also sequences where the amino acids have beenchemically modified, such as amino acid analogs, but which nonethelessretain substantially equivalent function. Functionally-equivalentvariants may occur as natural biological variations or may be preparedusing known techniques such as chemical synthesis, site-directedmutagenesis, random mutagenesis, or enzymatic cleavage and/or ligationof amino acids. Thus, modification of the amino-acid sequence to obtainvariant sequences may occur so long as the function of the polypeptideis not affected.

FIV functionally-equivalent variants within the scope of the inventionmay comprise conservatively substituted sequences, meaning that one ormore amino acid residues of the FIV polypeptide are replaced bydifferent residues that do not alter the secondary and/or tertiarystructure of the FIV polypeptide. Such substitutions may include thereplacement of an amino acid by a residue having similar physicochemicalproperties, such as charge density, size, configuration, orhypdrophilicity/hydrophobicity. For purposes of example only, suchsubstitutions could include substituting one aliphatic residue (Ile,Val, Leu, or Ala) for another, or substitution of basic residues Lys andArg, acidic residues Glu and Asp, amide residues Gln and Asn, hydroxylresidues Ser and Tyr, or aromatic residues Phe and Tyr. Conservativevariants can generally be identified by modifying a polypeptide sequenceof the invention and evaluating the antigenic activity of the modifiedpolypeptide using, for example, an immunohistochemical assay, anenzyme-linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), ora western blot assay. Further information regarding the making ofphenotypically silent amino acid exchanges may be found in Bowie et al.,Science 247:1306-1310 (1990).

Examples of functional equivalents of SEQ ID NO:1 are shown here with adescription of the various modifications to the peptides. SEQ IDDescription of NO: Sequence Amino Acid Changes 2 CELGSNQNQFFSK NativeFIV env sequence, amino acids 696 - 707, N-terminal C addition, C to Ssubstitutions 3 KVEAMEKFLYTAFAMQELGCN Native FTV env sequence,QNQFFCKIPLELWTR amino acids 680 - 715 4 TAFAMQELGSNQNQFFSK Native FIVenv sequence, amino acids 690 - 707, C to S substitutions 5YTAFAMQEWGCNQNQFFCA Native FIV env sequence, amino acids 689 - 707, L toW substitution, K to A substitution 6 TAFAMQELGCNQQQFFCA Native FIV envsequence, amino acids 690 - 707, N to Q, K to A substitutions 7YTAFAMQEIGCNQNQFFCA Native FIV env sequence, amino acids 689 - 707, L toI, K to A substitutions 8 CEGSNQNQFFSK Native FIV env sequence, aminoacids 696 - 707, N-terminal C addition; L deletion, C to S substitutions

Additional variants are also contemplated within the scope of theinvention, and such variants include amino and/or carboxyl terminalfusions, for example achieved by addition of amino acid sequences of anynumber of residues, as well as intrasequence insertion of one or moreamino acids. For example, amino acid sequences added may be thosederived from the whole or parts of other polypeptides or proteins, ormay be those provided in the corresponding positions in the FIV envelopeor viral protein. Longer peptides may comprise multiple copies of one ormore of the polypeptide sequences. Moreover, multiple copies of thepolypeptides may be coupled to a polyamino acid backbone, such as apolylysine backbone to form multiple antigen peptides (MAPs).

Deletional amino acid sequence variants are those in which one or moreamino acid residues are removed from the sequence. Insertional variantsexist when one or more amino acids are integrated into a predeterminedsite in the protein, although random insertion is an option withsuitable screening of the resulting product. In all cases, these andother FIV variants used retain substantially the same antigenicity ofthe FIV polypeptides. Other variants are also contemplated, includingthose where the amino acid substitutions are made in the area outsidethe antibody recognition regions of the protein. Fusion proteinscomprising two or more polypeptide sequences of FIV are also within thescope of the invention provided the sequences provide the appropriateantigenicity. Such polypeptides will generally correspond to at leastone epitope or mimitope that is characteristic of FIV. Bycharacteristic, it is meant that the epitope or mimitope will allowimmunologic detection of antibody directed to FIV in a physiologicalsample with reasonable assurance. Usually, it will be desirable that theepitope or mimitope, variant or fusion protein be immunologicallydistinct from (i.e., not cross-reactive with antibodies which recognize)viruses other than FIV.

An antigenically active variant differs by about, for example, 1, 2, 3,4, 5, or 6 amino acid residues from SEQ ID NO: 1, such as those shown inSEQ ID NOS: 2-8, or a fragment thereof. Where this comparison requiresalignment the sequences are aligned for maximum homology. Deletions,insertions, substitutions, repeats, inversions or mismatches areconsidered differences. The differences are, preferably, differences orchanges at a non-essential residue or a conservative substitution. Thesite of variation can occur anywhere in the polypeptide, as long as theresulting variant polypeptide is antigenically substantially similar toSEQ ID NO: 1, such as, for example, the variations shown in SEQ ID NOS:2-8 (see Tables 2 and 3). Exemplary functionally-equivalent variantsinclude those displaying 50% or more amino acid homology. Preferably,such homology is 60%, 70%, or greater than 80%. However, such variantsmay display a smaller percentage of homology overall and still fallwithin the scope of the invention where they have conserved regions ofhomology.

In some cases, one or more cysteine residues may be added to the terminiof the polypeptides in order to facilitate specific carrier linkage orto permit disulphide bonding to mimic antigenic loops and thus increasethe antigenicity. Moreover, a fatty acid or hydrophobic tail may beadded to the peptides to facilitate incorporation into delivery vehiclesand to increase antigenicity.

The FIV polypeptides used as detection reagents may be natural, i.e.,including the entire FIV protein or fragments thereof isolated from anatural source, or may be synthetic. The natural proteins may beisolated from the whole FIV virus by conventional techniques, such asaffinity chromatography. Polyclonal or monoclonal antibodies may be usedto prepare a suitable affinity column by well-known techniques.

Proteins that are immunologically cross-reactive with a natural FIVprotein can be chemically synthesized. For example, polypeptides havingfewer than about 100 amino acids, more usually fewer than about 80 aminoacids, and typically fewer than about 50 amino acids, may be synthesizedby the well-known Merrifield solid-phase synthesis method where aminoacids are sequentially added to a growing chain. (Merrifield, 1963, J.Am. Chem. Soc., 85:2149-2156). Recombinant proteins can also be used.These proteins may be produced by expression in cultured cells ofrecombinant DNA molecules encoding a desired portion of the FIV genome.The portion of the FIV genome may itself be natural or synthetic, withnatural genes obtainable from the isolated virus by conventionaltechniques. Of course, the genome of FIV is RNA, and it will benecessary to transcribe the natural RNA into DNA by conventionaltechniques employing reverse transcriptase. Polynucleotides may also besynthesized by well-known techniques. For example, short single-strandedDNA fragments may be prepared by the phosphoramidite method described byBeaucage and Carruthers, 1981, Tett. Letters 22:1859-1862.Double-stranded fragments may then be obtained either by synthesizingthe complementary strand and then annealing the strands together underappropriate conditions, or by adding the complementary strand using DNApolymerase with an appropriate primer sequence.

The natural or synthetic DNA fragments coding for the desired FIVprotein or fragment may be incorporated in a DNA construct capable ofintroduction to and expression in in vitro cell culture. Usually, theDNA constructs will be suitable for replication in a unicellular host,such as yeast or bacteria. They may also be intended for introductionand integration within the genome of cultured mammalian or othereukaryotic cells. DNA constructs prepared for introduction into bacteriaor yeast will include a replication system recognized by the host, theFIV DNA fragment encoding the desired polypeptide product,transcriptional and translational initiation regulatory sequences joinedto the 5′-end of the FIV DNA termination regulatory sequences joined tothe 3′-end of the fragment. The transcriptional regulatory sequenceswill include a heterologous promoter that is recognized by the host.Conveniently, a variety of suitable expression vectors are commerciallyavailable for a number of hosts.

To be useful in the detection methods of the present invention, thepolypeptides are obtained in a substantially pure form, that is,typically from about 50% w/w or more purity, substantially free ofinterfering proteins and contaminants. Preferably, the FIV polypeptidesare isolated or synthesized in a purity of at least 80% w/w, and morepreferably, in at least about 95% w/w purity. Using conventional proteinpurification techniques, homogeneous polypeptide compositions of atleast about 99% w/w purity can be obtained. For example, the proteinsmay be purified by use of the antibodies described hereinafter using theimmunoabsorbant affinity columns described hereinabove.

The method of the invention may be accomplished using immunoassaytechniques well known to those of skill in the art, including, but notlimited to, using microplates and lateral flow devices. In oneembodiment, an FIV protein is immobilized on a solid support at adistinct location. Detection of protein-antibody complexes on the solidsupport can be by any means known in the art. For example, U.S. Pat. No.5,726,010, which is incorporated herein by reference in its entirety,describes an example of a lateral flow device, the SNAP® immunoassaydevice (IDEXX Laboratories), useful in the present invention. Colloidalparticle based tests can also be used, such as the commerciallyavailable WITNESS® FIV diagnostic test (Synbiotics Corporation, Lyon,France).

Immobilization of one or more analyte capture reagents, e.g., FIVproteins, onto a device or solid support is performed so that an analytecapture reagent will not be washed away by the sample, diluent and/orwash procedures. One or more analyte capture reagents can be attached toa surface by physical adsorption (i.e., without the use of chemicallinkers) or by chemical binding (i.e., with the use of chemicallinkers). Chemical binding can generate stronger attachment of specificbinding substances on a surface and provide defined orientation andconformation of the surface-bound molecules.

Another embodiment of the invention provides a device that is suitablefor a lateral flow assay. For example, a test sample is added to a flowmatrix at a first region (a sample application zone). The test sample iscarried in a fluid flow path by capillary action to a second region ofthe flow matrix where a label capable of binding and forming a firstcomplex with an analyte in the test sample. The first complex is carriedto a third region of the flow matrix where an FIV protein is immobilizedat a distinct location. A second complex is formed between animmobilized protein and the first complex including the antibody fromthe sample. For example, a first complex comprising a gold sol particleand an FIV protein bound to an FIV antibody will specifically bind andform a second complex with a second immobilized FIV protein or with asecond antibody directed to feline antibodies. The label that is part ofthe second complex can be directly visualized.

In another aspect, the invention includes one or more labeled specificbinding reagents that can be mixed with a test sample prior toapplication to a device for of the invention. In this case it is notnecessary to have labeled specific binding reagents deposited and driedon a specific binding reagent pad in the device. A labeled specificbinding reagent, whether added to a test sample or pre-deposited on thedevice, can be for example, a labeled FIV protein that specificallybinds an antibody for FIV.

Any or all of the above embodiments can be provided as a kit. In oneparticular example, such a kit would include a device complete withspecific binding reagents (e.g., a non-immobilized labeled specificbinding reagent and an immobilized analyte capture reagent) and washreagent, as well as detector reagent and positive and negative controlreagents, if desired or appropriate. In addition, other additives can beincluded, such as stabilizers, buffers, and the like. The relativeamounts of the various reagents can be varied, to provide forconcentrations in solution of the reagents that substantially optimizethe sensitivity of the assay. Particularly, the reagents can be providedas dry powders, usually lyophilized, which on dissolution will providefor a reagent solution having the appropriate concentrations forcombining with a sample.

An FIV protein can be an immobilized analyte capture reagent in areaction zone (solid phase). A second analyte capture reagent, i.e. asecond FIV protein, that has been conjugated to a label, can either beadded to the sample before the sample is added to the device, or thesecond analyte capture reagent can be incorporated into the device. Forexample the labeled specific binding reagent can be deposited and driedon a fluid flow path that provides fluid communication between thesample application zone and the solid phase. Contact of the labeledspecific binding reagent with the fluid sample results in dissolution ofthe labeled specific binging reagent.

The device may also include a liquid reagent that transports unboundmaterial (e.g., unreacted fluid sample and unbound specific bindingreagents) away from the reaction zone (solid phase). A liquid reagentcan be a wash reagent and serve only to remove unbound material from thereaction zone, or it can include a detector reagent and serve to bothremove unbound material and facilitate analyte detection. For example,in the case of a specific binding reagent conjugated to an enzyme, thedetector reagent includes a substrate that produces a detectable signalupon reaction with the enzyme-antibody conjugate at the reactive zone.In the case of a labeled specific binding reagent conjugated to aradioactive, fluorescent, or light-absorbing molecule, the detectorreagent acts merely as a wash solution facilitating detection of complexformation at the reactive zone by washing away unbound labeled reagent.

Two or more liquid reagents can be present in a device, for example, adevice can comprise a liquid reagent that acts as a wash reagent and aliquid reagent that acts as a detector reagent and facilitates analytedetection.

A liquid reagent can further include a limited quantity of an“inhibitor”, i.e., a substance that blocks the development of thedetectable end product. A limited quantity is an amount of inhibitorsufficient to block end product development until most or all excess,unbound material is transported away from the second region, at whichtime detectable end product is produced.

The following are provided for exemplification purposes only and are notintended to limit the scope of the invention described in broad termsabove. All references cited in this disclosure are incorporated hereinby reference.

EXAMPLES Example 1

Eight cats testing negative for FIV with the SNAP® FeLV Ag/FIV Ab testkits were vaccinated with Fel-O-Vax® FIV vaccine, Fort Dodge AnimalHealth, Fort Dodge Iowa. This vaccine is produced from multiple strainsof the whole killed FIV virus. The cats were vaccinated following themanufacturer's directions at day 0, 14, and 28. Two cats testingnegative for FIV were not vaccinated and were included as controls forthis study.

Blood samples were obtained from each of the ten cats in the vaccinationstudy at day zero and every seven days for 12 weeks and stored frozenuntil testing. In addition, blood samples from FIV negative cats andcats naturally infected with FIV, confirmed FIV Ab negative or positiveby a western immunoblot confirmatory test, were also tested.

Sample testing was accomplished using a SNAP® ELISA format. SNAP® devicetechnology was used to provide a solid phase with reversible,chromatographic flow of sample, and automatic, sequential flow of washand enzyme substrate solutions as described in U.S. Pat. No. 5,726,010.

For the SNAP® device, FIV gag p24 (recombinant) and an FIV env 696-707with additional N-terminal cysteine—CELGCNQNQFFCK [SEQ ID NO:1]—proteinswere deposited to form a single antibody capture spot on the solidphase. A negative control reagent was deposited to form a negativecontrol spot and a positive control reagent was deposited to form apositive control spot on the solid phase of the SNAP® device. The gag orenv proteins were chemically conjugated to the enzyme horseradishperoxidase and provided in a solution consisting of a buffer, detergent,and animal serum components.

Serum samples were combined with either gag or env protein-enzymeconjugate solution, and applied to the SNAP® device. Following a shortincubation period, the device was activated. Color development on thepositive control spot indicated the test was valid. Color development onthe sample spot greater than color development on the negative controlspot indicated the presence of FIV antibody in the sample and was scoredas a positive test result. Test results were determined visually and areshown in Table 1. TABLE 1 gag Ab env Ab test result test result AnimalID Status Day (visual) (visual) NV1 not vaccinated, not infected 0 NEGNEG NV1 not vaccinated, not infected 7 NEG NEG NV1 not vaccinated, notinfected 14 NEG NEG NV1 not vaccinated, not infected 21 NEG NEG NV1 notvaccinated, not infected 28 NEG NEG NV1 not vaccinated, not infected 35NEG NEG NV1 not vaccinated, not infected 42 NEG NEG NV1 not vaccinated,not infected 49 NEG NEG NV1 not vaccinated, not infected 56 NEG NEG NV1not vaccinated, not infected 63 NEG NEG NV1 not vaccinated, not infected70 NEG NEG NV1 not vaccinated, not infected 77 NEG NEG NV1 notvaccinated, not infected 84 NEG NEG NV2 not vaccinated, not infected 0NEG NEG NV2 not vaccinated, not infected 7 NEG NEG NV2 not vaccinated,not infected 14 NEG NEG NV2 not vaccinated, not infected 21 NEG NEG NV2not vaccinated, not infected 28 NEG NEG NV2 not vaccinated, not infected35 NEG NEG NV2 not vaccinated, not infected 42 NEG NEG NV2 notvaccinated, not infected 49 NEG NEG NV2 not vaccinated, not infected 56NEG NEG NV2 not vaccinated, not infected 63 NEG NEG NV2 not vaccinated,not infected 70 NEG NEG NV2 not vaccinated, not infected 77 NEG NEG NV2not vaccinated, not infected 84 NEG NEG V1 vaccinated, not infected 0NEG NEG V1 vaccinated, not infected 7 NEG NEG V1 vaccinated, notinfected 14 NEG NEG V1 vaccinated, not infected 21 POS NEG V1vaccinated, not infected 28 POS NEG V1 vaccinated, not infected 35 POSPOS V1 vaccinated, not infected 42 POS NEG V1 vaccinated, not infected49 POS NEG V1 vaccinated, not infected 56 POS NEG V1 vaccinated, notinfected 63 POS NEG V1 vaccinated, not infected 70 POS NEG V1vaccinated, not infected 77 POS NEG V1 vaccinated, not infected 84 POSNEG V2 vaccinated, not infected 0 NEG NEG V2 vaccinated, not infected 7NEG NEG V2 vaccinated, not infected 14 NEG NEG V2 vaccinated, notinfected 21 NEG NEG V2 vaccinated, not infected 28 NEG NEG V2vaccinated, not infected 35 POS NEG V2 vaccinated, not infected 42 POSNEG V2 vaccinated, not infected 49 POS NEG V2 vaccinated, not infected56 POS NEG V2 vaccinated, not infected 63 POS NEG V2 vaccinated, notinfected 70 POS NEG V2 vaccinated, not infected 77 POS NEG V2vaccinated, not infected 84 POS NEG V3 vaccinated, not infected 0 NEGNEG V3 vaccinated, not infected 7 NEG NEG V3 vaccinated, not infected 14NEG NEG V3 vaccinated, not infected 21 NEG NEG V3 vaccinated, notinfected 28 NEG NEG V3 vaccinated, not infected 35 POS NEG V3vaccinated, not infected 42 POS NEG V3 vaccinated, not infected 49 POSNEG V3 vaccinated, not infected 56 POS NEG V3 vaccinated, not infected63 POS NEG V3 vaccinated, not infected 70 POS NEG V3 vaccinated, notinfected 77 POS NEG V3 vaccinated, not infected 84 POS NEG V4vaccinated, not infected 0 NEG NEG V4 vaccinated, not infected 7 NEG NEGV4 vaccinated, not infected 14 POS NEG V4 vaccinated, not infected 21POS NEG V4 vaccinated, not infected 28 POS NEG V4 vaccinated, notinfected 35 POS NEG V4 vaccinated, not infected 42 POS NEG V4vaccinated, not infected 49 POS NEG V4 vaccinated, not infected 56 POSNEG V4 vaccinated, not infected 63 POS NEG V4 vaccinated, not infected70 POS NEG V4 vaccinated, not infected 77 POS NEG V4 vaccinated, notinfected 84 POS NEG V5 vaccinated, not infected 0 NEG NEG V5 vaccinated,not infected 7 NEG NEG V5 vaccinated, not infected 14 NEG NEG V5vaccinated, not infected 21 POS POS V5 vaccinated, not infected 28 POSNEG V5 vaccinated, not infected 35 POS NEG V5 vaccinated, not infected42 POS NEG V5 vaccinated, not infected 49 POS NEG V5 vaccinated, notinfected 56 POS NEG V5 vaccinated, not infected 63 POS NEG V5vaccinated, not infected 70 POS NEG V5 vaccinated, not infected 77 POSNEG V5 vaccinated, not infected 84 POS NEG V5 vaccinated, not infected 0NEG NEG V5 vaccinated, not infected 7 NEG NEG V5 vaccinated, notinfected 14 NEG NEG V5 vaccinated, not infected 21 POS NEG V5vaccinated, not infected 28 POS NEG V5 vaccinated, not infected 35 POSPOS V5 vaccinated, not infected 42 POS NEG V5 vaccinated, not infected49 POS NEG V5 vaccinated, not infected 56 POS NEG V5 vaccinated, notinfected 63 POS NEG V5 vaccinated, not infected 70 POS NEG V5vaccinated, not infected 77 POS NEG V5 vaccinated, not infected 84 POSNEG V7 vaccinated, not infected 0 NEG NEG V7 vaccinated, not infected 7NEG NEG V7 vaccinated, not infected 14 NEG NEG V7 vaccinated, notinfected 21 POS NEG V7 vaccinated, not infected 28 POS NEG V7vaccinated, not infected 35 POS NEG V7 vaccinated, not infected 42 POSPOS V7 vaccinated, not infected 49 POS POS V7 vaccinated, not infected56 POS NEG V7 vaccinated, not infected 63 POS NEG V7 vaccinated, notinfected 70 POS NEG V7 vaccinated, not infected 77 POS NEG V7vaccinated, not infected 84 POS NEG V8 vaccinated, not infected 0 NEGNEG V8 vaccinated, not infected 7 NEG NEG V8 vaccinated, not infected 14POS NEG V8 vaccinated, not infected 21 POS NEG V8 Vaccinated, notinfected 28 POS NEG V8 Vaccinated, not infected 35 POS NEG V8Vaccinated, not infected 42 POS NEG V8 Vaccinated, not infected 49 POSNEG V8 Vaccinated, not infected 56 POS NEG V8 Vaccinated, not infected63 POS NEG V8 Vaccinated, not infected 70 POS NEG V8 Vaccinated, notinfected 77 POS NEG V8 Vaccinated, not infected 84 POS NEG Inf1 Notvaccinated, infected ND POS POS Inf2 Not vaccinated, infected ND POS POSInf3 Not vaccinated, infected ND POS POS Inf4 Not vaccinated, infectedND POS POS Inf5 Not vaccinated, infected ND POS POS Inf6 Not vaccinated,infected ND POS POS Inf7 Not vaccinated, infected ND POS POS Inf8 Notvaccinated, infected ND POS POS Inf9 Not vaccinated, infected ND POS POSinf10 Not vaccinated, infected ND POS POS

Example 2

Microplate ELISA analysis was performed on serum samples collected fromconfirmed FIV negative and infected cats, and cats vaccinated with theFEL-O-VAX® FIV vaccine in an indirect assay format with individual FIVpolypeptides on the solid phase and anti-(feline IgG) peroxidaseconjugate. Antibodies to env FIV proteins were detected using thesepeptides as antigen reagents: CELGCNQNQFFCK [SEQ ID NO:1] CELGSNQNQFFSK[SEQ ID NO:2]

The polypeptides were synthesized using a commercial instrument andfollowing the manufacturer's instructions. Polypeptide stocks wereprepared at 5 mg/ml in DMSO. The polypeptides were then coated onmicroplate wells (peptide @ 10 ug/ml in 50 mM Tris-HCl pH 7.4, 100ul/well). The plates were then blocked/overcoated with 2% Tween-20/2.5%sucrose, allowed to dry in mylar bags with desiccant.

For the assays, feline serum samples (100 ul/well, diluted 1/1000 in 50%fetal bovine serum) were added to the wells and the plates wereincubated for ten minutes at room temperature. Following incubation, themicroplates were washed with PBS/Tween. Goat Anti-(cat IgG):peroxidaseconjugate was added to the wells (100 ul/well anti-catIgG:peroxidasediluted in 50% fetal bovine serum). The plates were incubated foranother fifteen minutes at room temperature and washed a second timewith PBS/Tween. Peroxidase substrate was added (100 ul/well, tetramethylbenzidine peroxidase substrate) and the plates were incubated a thirdtime for ten minutes at room temperature. A hydrofluoric acid stopsolution (50 ul/well) was added to the plates. Sample antibody bindingwas measured by determining peroxidase activity (colored product) with aspectrophotometer (A650 nm). Significant, substantial antibody bindingfor a sample is considered to be A650 nm greater than 0.200. The IDEXXPetChek® Anti-FIV antibody test kit was also run on these samples as areference test. The results are shown in Table 2. TABLE 2 FIV infected,not vaccinated: PetChek ® seq ID 1 seq ID 2 Anti-FIV Sample A(650 nm)A(650 nm) result 2689:44 7 1.862 0.908 positive 194 1.305 1.327 positive197 1.905 0.687 positive 22488 1.943 0.802 positive F9-1525 Dec. 5, 19991.467 1.163 positive 23804 255 1.884 0.991 positive F0-138 Jan. 23, 20001.938 1.409 positive 23430 244 1.799 0.786 positive 56360 60 1.901 1.391positive 57561 181 1.882 1.782 positive 56897 187 1.738 0.813 positive21518 1.408 1.000 positive 58178 232 1.845 1.386 positive 23805 2531.481 1.005 positive 21636 1.905 1.726 positive 23119 1.546 0.888positive 55214 190 1.570 1.214 positive 57601 215 1.373 0.725 positive21583 0.593 0.408 positive F9-881 1.497 0.926 positive 58036 224 1.7211.387 positive F0-162 Feb. 13, 2000 1.360 1.068 positive 57157 141 0.9360.603 positive 23321 211 1.339 1.183 positive 56035 1 0.925 0.620positive mean 1.565 1.048 seq ID 1 seq ID 2 PetChek sample A(650 nm)A(650 nm) result FIV negative, vaccinated: Vx 3495 Day 84 0.040 0.037positive Vx SK4 Day 84 0.043 0.038 positive Vx 3532 Day 84 0.044 0.034positive Vx 3528 Day 84 0.033 0.035 positive Vx 3519 Day 84 0.037 0.035positive Vx 3517 Day 84 0.050 0.034 positive Vx SK4 Day 35 0.115 0.056positive Vx C3532 Day 42 0.101 0.060 positive Vx C3519 Day 35 0.0900.044 positive Vx SK4 Day 21 0.052 0.035 positive Vx C3517 Day 35 0.1100.082 positive mean 0.065 0.045 FIV negative, not vaccinated: 57360 1770.033 0.037 negative 57551 205 0.032 0.031 negative F9-1375 0.033 0.041negative 57208 146 0.037 0.034 negative F9-1519 Dec. 5, 1999 0.050 0.032negative 57435 272 8/17 0.036 0.039 negative 57975 236 0.031 0.035negative 57323 174 0.035 0.046 negative 56728 200 0.033 0.039 negativeF9-1191 0.034 0.027 negative 57528 197 0.034 0.033 negative 57095 1250.034 0.034 negative 56704 154 0.033 0.033 negative 57911 235 0.0340.040 negative F9-1455 Nov. 14, 1999 0.034 0.034 negative F0-53 Jan. 23,2000 0.033 0.035 negative 57222 153 0.033 0.036 negative 56746 226 0.0320.036 negative 57238 0.030 0.035 negative 2873 0.032 0.034 negative22151 80 0.033 0.026 negative 57611 216 0.031 0.035 negative 57211 1470.034 0.035 negative F9-1211 0.034 0.033 negative 58203 230 0.031 0.033negative 57203 145 0.033 0.032 negative mean 0.034 0.035

Example 3

Microplate ELISA analysis was performed as in Example 2 on serum samplescollected from confirmed FIV negative and infected cats, and catsvaccinated with the FEL-O-VAX® FIV vaccine. Antibodies to FIV env weredetected using these peptides as antigen reagents: CELGCNQNQFFCK [SEQ IDNO:1] KVEAMEKFLYTAFAMQELGCNQNQFFCKIPLELWTR [SEQ ID NO:3]TAFAMQELGSNQNQFFSK [SEQ ID NO:4] YTAFAMQEWGCNQNQFFCA [SEQ ID NO:5]TAFAMQELGCNQQQFFCA [SEQ ID NO:6] YTAFAMQEIGCNQNQFFCA [SEQ ID NO:7]CEGSNQNQFFSK [SEQ ID NO:8]

Significant, substantial antibody binding for a sample is considered tobe A650 nm greater than 0.200). The results are reported in Table 3 and4. TABLE 3 seq ID 1 seq ID 3 seq ID 4 seq ID 5 seq ID 6 seq ID 7 PetCheksample A(650 nm) A(650 nm) A(650 nm) A(650 nm) A(650 nm) A(650 nm)result FIV infected, not vaccinated: 2689:44 7 1.862 1.911 0.908 1.6960.893 1.153 positive 194 1.305 1.845 1.327 0.636 0.948 0.523 positive197 1.905 1.888 0.687 1.623 1.590 1.027 positive 22488 1.943 1.714 0.8021.162 1.663 1.296 positive 23804 255 1.884 1.115 0.991 0.829 1.078 1.036positive 24034 283 1.508 1.423 0.663 1.436 1.318 1.202 positive F0-138Jan. 23, 2000 1.938 1.922 1.409 1.835 1.412 1.548 positive 56360 601.901 2.808 1.391 1.553 1.270 1.298 positive 57561 181 1.882 1.799 1.7821.747 1.000 1.293 positive 56897 187 1.738 1.900 0.813 1.492 0.929 1.102positive 21518 1.408 1.319 1.000 1.419 1.032 0.955 positive 58178 2321.845 1.952 1.386 1.338 1.392 1.237 positive 23805 253 1.481 1.205 1.0050.562 0.721 0.599 positive 21636 1.905 1.600 1.726 1.225 1.811 1.238positive 58232 242 1.221 1.005 0.454 1.580 0.686 1.297 positive 231191.546 1.446 0.888 1.154 0.447 1.156 positive 57601 215 1.373 0.985 0.7250.987 0.722 0.601 positive F9-881 1.497 1.951 0.926 0.587 1.272 0.589positive 23938 323 1.489 1.203 0.635 1.475 0.999 1.333 positive 58036224 1.721 1.855 1.387 1.218 0.828 1.102 positive 22879 1.332 1.602 0.4681.117 0.752 1.055 positive F0-162 Feb. 13, 2000 1.360 1.118 1.068 1.2840.702 1.046 positive 57157 141 0.936 1.089 0.603 0.491 0.415 0.417positive 23321 211 1.339 1.057 1.183 0.751 1.308 0.886 positive 56035 10.925 0.776 0.620 0.368 0.691 0.508 positive mean 1.570 1.540 0.9941.183 1.035 1.020 FIV negative, vaccinated: Vx C3520 Day 35 0.234 0.1990.058 0.142 0.065 0.116 positive Vx C3511 Day 49 0.208 0.115 0.155 0.2090.111 0.180 positive Vx C3519 Day 35 0.062 0.055 0.053 0.091 0.058 0.055positive Vx C3517 Day 35 0.110 0.098 0.082 0.093 0.063 0.074 positive VxSK4 Day 21 0.052 0.045 0.035 0.082 0.038 0.044 positive mean 0.133 0.1020.077 0.123 0.067 0.094 FIV negative, not vaccinated: 57975 236 0.0310.040 0.035 0.040 0.043 0.036 negative 57323 174 0.035 0.039 0.046 0.0320.042 0.034 negative 56728 200 0.033 0.035 0.039 0.049 0.044 0.042negative 56956 209 0.036 0.036 0.035 0.049 0.038 0.036 negative F9-1638143119 0.039 0.041 0.035 0.037 0.036 0.039 negative F9-1191 0.034 0.0510.027 0.040 0.037 0.037 negative 57528 197 0.034 0.038 0.033 0.039 0.0350.035 negative 57095 125 0.034 0.036 0.034 0.038 0.035 0.035 negative56704 154 0.033 0.036 0.033 0.040 0.027 0.035 negative 57911 235 0.0340.035 0.040 0.056 0.050 0.038 negative F9-1455 Nov. 14, 1999 0.034 0.0330.034 0.037 0.037 0.033 negative F0-53 Jan. 23, 2000 0.033 0.041 0.0350.038 0.037 0.036 negative 57222 153 0.033 0.033 0.036 0.041 0.037 0.036negative 56746 226 0.032 0.035 0.036 0.038 0.036 0.034 negative F9-12780.034 0.035 0.024 0.038 0.036 0.034 negative 57238 0.030 0.033 0.0350.037 0.036 0.035 negative 2873 0.032 0.040 0.034 0.036 0.033 0.034negative 22151 80 0.033 0.032 0.026 0.039 0.036 0.047 negative 57611 2160.031 0.031 0.035 0.067 0.043 0.044 negative mean 0.033 0.037 0.0340.042 0.038 0.044

TABLE 4 seq ID 1 seq ID 8 PetChek sample A(650 nm) A(650 nm) result FIVinfected, not vaccinated: 2426-91A 1.170 0.838 positive 2426-64-21 0.7540.654 positive 2605 1.679 0.965 positive 21636 1.916 1.918 positive 26141.544 1.095 positive 3364-89 0.375 0.677 positive 58376-274 1.425 1.370positive Gonzalez 1.622 1.920 positive Butzi 0.627 0.902 positiveStanley 0.990 0.442 positive JL-60 1.651 2.127 positive mean 1.250 1.173FIV negative, vaccinated: Vx 3520 Day 84 0.040 0.040 positive Vx 3519Day 84 0.036 0.041 positive Vx 3532 Day 84 0.037 0.042 positive Vx SK4Day 84 0.038 0.048 positive Vx G1 week 4 0.092 0.092 positive Vx G1 week5 0.085 0.101 positive Vx G1 week 6 0.091 0.111 positive Vx G1 week 70.093 0.103 positive Vx G1 week 8 0.074 0.096 positive Vx G1 week 120.070 0.085 positive mean 0.066 0.076 FIV negative, not vaccinated:2483-83-23 0.034 0.035 negative 2483-83-30 0.035 0.035 negative2483-83-33 0.034 0.035 negative 2151-05H 0.037 0.049 negative 7685470.036 0.034 negative 769703 0.035 0.035 negative 768513 0.037 0.036negative F6263E 0.034 0.058 negative 2377-1-38 0.037 0.034 negative14834 0.036 0.036 negative 14151 0.035 0.034 negative D1606315 0.0340.035 negative mean 0.035 0.038

Although various specific embodiments of the present invention have beendescribed herein, it is to be understood that the invention is notlimited to those precise embodiments and that various changes ormodifications can be affected therein by one skilled in the art withoutdeparting from the scope and spirit of the invention.

1. A polypeptide selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
 2. A method of distinguishing between animals that have been naturally infected with FIV and animals that have not been infected or have been vaccinated against an FIV infection, the method comprising: contacting a biological sample from an animal with a polypeptide of SEQ ID NO:1 or claim 1; detecting whether FIV antibodies in the sample substantially bind to the polypeptide; determining that the animal is naturally infected by detecting that antibodies in the sample substantially bind to the FIV polypeptide and determining that the animal has been vaccinated or not infected by detecting that antibodies in the sample do not substantially bind to the FIV polypeptide.
 3. The method of claim 2 wherein the biological sample is obtained from an animal that has not received an FIV vaccine within about the prior 12 weeks.
 4. A method of determining whether a felid has been vaccinated against FIV or is naturally infected with FIV, the method comprising: (a) detecting, before a period a time following vaccination sufficient for antibodies that are not a significant component of the animal's immune response to an FIV vaccine to be substantially eliminated from the felid, whether the felid has antibodies against an FIV peptide of SEQ ID NO:1 or claim 1; (b) detecting, after a period a time following vaccination sufficient for antibodies that are not a significant component of an animal's immune response to the vaccine to be substantially eliminated from the felid, whether the felid has antibodies against the FIV polypeptide; (c) determining that the felid has been successfully vaccinated by detecting antibodies in step a but not in step b, and (d) determining that the felid is naturally infected by detecting antibodies in steps a and b.
 5. A method of determining whether a felid has not been infected by FIV or has been vaccinated against FIV, the method comprising: analyzing a biological sample from the felid to detect the presence or absence of antibodies against an FIV peptide of SEQ ID NO:1 or claim 1; and determining that the animal has not been infected or has been or vaccinated by determining the absence of such antibodies.
 6. A method of determining whether or not a felid has been vaccinated against FIV or naturally infected with FIV comprising: providing a test device comprising a polypeptide of SEQ ID NO:1 or claim 1; obtaining a biological sample from a felid; running the biological sample on the test device so that the sample contacts the polypeptide; and reading the test device, wherein a positive result indicates the cat has been naturally infected with FIV or vaccinated against FIV and wherein a negative result indicates the cat has not been naturally infected with FIV and not vaccinated against FIV.
 7. A method of determining the origin of FIV polypeptide in a biological sample from an animal suspected of having a natural FIV infection or having been vaccinated against FIV, the method comprising: obtaining a biological sample from an animal that has not been vaccinated with an FIV vaccine within about the prior twelve weeks, contacting the biological sample obtained from the animal with: (a) a first polypeptide that substantially binds with a significant component of the animal's antibody response to the FIV vaccine, and (b) a polypeptide of SEQ ID NO:1 or claim 1, detecting whether antibodies in the sample substantially bind to one or both the polypeptides; and determining that the animal is naturally infected when the antibodies substantially bind to both peptides (a) and (b), and determining that the animal has been vaccinated when the antibodies bind to only peptide (a).
 8. The method of claim 7 wherein the first polypeptide comprises FIV p15.
 9. The method of claim 7 wherein the first polypeptide comprises FIV p24.
 10. A diagnostic device comprising: a dry porous carrier; a first detection reagent immobilized on the porous carrier, said first detection reagent comprising a protein that captures FIV antibodies generated by an animal in response to either a natural FIV infection or an FIV vaccination; and a second detection reagent immobilized on the porous carrier, said second detection reagent comprising the polypeptide of SEQ ID NO:1 or claim
 1. 11. The diagnostic device according to claim 10, wherein the first detection reagent comprises FIV p15.
 12. The diagnostic device according to claim 10, wherein the first detection reagent comprises FIV p24.
 13. A method for determining whether a felid has been vaccinated against FIV or is naturally infected with FIV, the method comprising contacting a biological sample with the diagnostic device of claim 10 and detecting whether antibodies in the sample substantially bind one or both of the first detection reagent and the second detection reagent.
 14. A diagnostic device comprising: a first porous carrier comprising a first detection reagent immobilized thereon; said first detection reagent comprising a polypeptide that captures FIV antibodies generated by an animal in response to either a natural FIV infection or an FIV vaccination; and a second porous carrier comprising a second detection reagent immobilized thereon, said second detection reagent comprising a polypeptide of SEQ ID NO:1 or claim
 1. 15. The diagnostic device according to claim 14, wherein the first detection reagent comprises FIV p15.
 16. The diagnostic device according to claim 14, wherein the first detection reagent comprises FIV p24.
 17. A method of determining the vaccination status of a felid comprising: providing the polypeptide of SEQ ID NO:1 or claim 1; contacting a biological sample from a felid with the polypeptide to form a polypeptide/antibody complex; and determining the presence or absence of the polypeptide/antibody complex, wherein the presence of the complex represents natural infection and the absence of the complex represents either vaccination or no infection.
 18. The method of claim 17 wherein the biological sample is obtained from an animal that has not received an FIV vaccine within about the prior 12 weeks.
 19. A method of determining whether a felid has been vaccinated against FIV or is naturally infected with FIV comprising determining the felid's immune response to the polypeptide of SEQ ID NO:1 or claim
 1. 20. Use of a polypeptide derived from FIV env in the manufacture of a kit for the diagnosis of whether a cat has been vaccinated against FIV.
 21. Use, as claimed in claim 20, wherein the method also involves determining whether the cat has been infected with FIV.
 22. The polypeptide as claimed in claim 1, wherein the polypeptide comprises antigenic fragments and functionally-equivalents thereof.
 23. The polypeptide as claimed in claim 1, wherein the polypeptide is in the form of a fusion protein comprising an additional polypeptide fused to said amino acid sequence, or which is coupled to a carrier protein or polypeptide. 